The purpose of this career development grant is to establish myself as an independent biomedical scientist who develops tissue regeneration therapies through study of skeletal development and disease in engineered microenvironment models. The ultimate goal of the research is to tissue engineer a growth plate (GP) to treat skeletal dysplasia and complex bone injuries. The GP is the cartilaginous structure at the ends of limb bones that drives lengthwise growth. To restore limb length and geometry in patients with GP and compromised facial and extremity bone injuries, patients are often subject to distraction osteogenesis, a burdensome and prolonged (3-6 months) procedure using external fixators that pierce the skin, cause great pain, and risk infection and scaring. Bone regeneration and lengthening using a bone marrow stem cell (MSC) derived tissue engineered GP may prove less invasive and more effective than autografts and acellular therapies through its ability to resist hypoxia and recruit blood vessels. The growth factors parathyroid hormone related peptide (PTHrP) and indian hedgehog (IHH) are major effectors of GP development via a negative feedback gradient loop. It is unknown if PTHrP and IHH gradients are sufficient to generate GP zonal structure. We hypothesize that counter gradients of PTHrP and IHH agonists across a hydrogel construct seeded with MSC-derived chondrocytes will induce cell differentiation into a GP spatial architecture. We will create a novel model using microfluidic bioreactors and photopatterning of cells and hydrogels. Aim 1 is to design and generate model morphogen gradient profiles in hydrogels. We will generate uniaxial gradients of fluorescently tagged idealized morphogens across poly(ethylene glycol) diacrylate (PEGDA) gels housed within our bioreactor. Aim 2 is to investigate growth GP-like zonal differentiation of chondrocytes in generated constructs versus assembled constructs in vitro. We will prepare MSC-derived chondrocytes with culture in supplemented chondrogenic medium. To create generated constructs, we will isolate and embed these chondrocytes in PEGDA gels, and subject gels to a PTHrP and IHH agonist counter-gradient and individual gradients. To create assembled constructs, we will pre-differentiate chondrocytes into reserve and hypertrophic populations with PTHrP and IHH agonist supplemented cultures, and then photopattern these at opposite ends of untreated cells forming a tri-layered gel. Aim 3 is to evaluate engineered GP development and integration with vasculature in vivo. We will implant these constructs in dorsal subcutaneous pockets of immunodeficient mice and use histological and micro-CT assays to evaluate construct growth and GP-like structure maintenance.